Adaptation mechanism of three Impatiens species to different habitats based on stem morphology, lignin and MYB4 gene

Background Impatiens is an important genus with rich species of garden plants, and its distribution is extremely extensive, which is reflected in its diverse ecological environment. However, the specific mechanisms of Impatiens’ adaptation to various environments and the mechanism related to lignin remain unclear. Results Three representative Impatiens species,Impatiens chlorosepala (wet, low degree of lignification), Impatiens uliginosa (aquatic, moderate degree of lignification) and Impatiens rubrostriata (terrestrial, high degree of lignification), were selected and analyzed for their anatomical structures, lignin content and composition, and lignin-related gene expression. There are significant differences in anatomical parameters among the stems of three Impatiens species, and the anatomical structure is consistent with the determination results of lignin content. Furthermore, the thickness of the xylem and cell walls, as well as the ratio of cell wall thickness to stem diameter have a strong correlation with lignin content. The anatomical structure and degree of lignification in Impatiens can be attributed to the plant's growth environment, morphology, and growth rate. Our analysis of lignin-related genes revealed a negative correlation between the MYB4 gene and lignin content. The MYB4 gene may control the lignin synthesis in Impatiens by controlling the structural genes involved in the lignin synthesis pathway, such as HCT, C3H, and COMT. Nonetheless, the regulation pathway differs between species of Impatiens. Conclusions This study demonstrated consistency between the stem anatomy of Impatiens and the results obtained from lignin content and composition analyses. It is speculated that MYB4 negatively regulates the lignin synthesis in the stems of three Impatiens species by regulating the expression of structural genes, and its regulation mechanism appears to vary across different Impatiens species. This study analyses the variations among different Impatiens plants in diverse habitats, and can guide further molecular investigations of lignin biosynthesis in Impatiens. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-024-05115-3.


Introduction
The emergence of lignin is one of the most important alterations in the evolution of aquatic plants into terrestrial environments, enabling them to adapt to the terrestrial ecological conditions.Lignin plays a crucial role in enhancing plants' mechanical strength, water transport and resistance to external bacteria invasion.In addition, it improves plants' defense capacity against biological and abiotic stresses [1][2][3][4].Lignin comprises a significant proportion of plant cell structure, contributing about a third of woody plant biomass on land [5].Lignin primarily consists of three monomers-p-hydroxyphenyl (H-lignin), guaiacyl (G-lignin) and syringyl lignin (S-lignin)-that are polymerized.Some atypical monomers such as catechyl and 5-hydroxyguaiacyl (5H) have also been detected [6,7].
The regulation of plant lignin synthesis is governed through a three-level network.NAC transcription factors serve as the first-order switches, AtMYB46 and AtMYB83 are the second-order switches [12][13][14].Additional MYB transcription factors, such as AtMYB20, AtMYB42, AtMYB43 and AtMYB85 are the third-order regulatory genes [15,16].These transcription factors have the capability to activate the expression of lignin synthesis genes which, in turn, further influences the formation of plant secondary cell walls.It has been reported that MYB transcription factors inhibiting lignin synthesis can be found in most plants, such as MYB4 in Arabidopsis thaliana [17,18], CmMYB8 in Chrysanthemum morifolium [19], and EgMYB1 in Eucalyptus gunnii [20], which can negatively regulate lignin synthesis.
The MYB4 transcription factor possesses R2 and R3 domains specific to the MYB family and belongs to the R2R3-MYB.Overexpression of AtMYB4 in Nicotiana tabacum resulted in decreased in tobacco growth and lignin content [17,21].Similarly, overexpression of PvMYB4 in Panicum virgatum led to a decrease in lignin content [22].Transgenic Arabidopsis thaliana plants that overexpress NnMYB4 exhibited a significant reduction in stem lignin content, suggesting that NnMYB4 from Nelumbo nucifera may function as a transcriptional inhibitor of lignin synthesis [23].Meanwhile, overexpressing BpMYB4 in Arabidopsis thaliana promoted stem growth while reducing lignin deposition, indicating that BpMYB4 inhibits lignin biosynthesis [24].It can be seen that the transcription factor MYB4 plays a negative role in regulating lignin in most plants.
The family Balsaminaceae contains of only two genera: single species of Hydrocera and Impatiens.Impatiens is mainly distributed in southwest China, especially in Yunnan.The Impatiens species are mostly herbaceous plants with rich colors, peculiar flower shapes, and fleshy stems.Most are annuals, though a few are perennials.Additionally, there are also shrub-like species, characterized by hard and lignified stems.Currently, the research of Impatiens species focuses on cultivation management, tissue culture, flower organ development, plant disease, molecular biology and evolution [25][26][27][28][29].For instance, Li [26] explored the related development mechanism of the spur of I. uliginosa with important ecological value.Janssens [30] reconstructed the phylogenetic tree of 85 Impatiens species using the chloroplast gene atpB-rbcL and the nuclear gene ITS and validated the sisterhood relationship between Impatiens and Hydrangea.
Impatiens species inhabit tropical and subtropical regions and typically thrive in high-humidity habitats, including forests and streams, while some species are adapted to grow in arid areas [31].Due to its complex and diverse habitats, the genus Impatiens has a great diversity, including rich ecotypes, such as herbs, subshrubs, woody plants [32] and erect or creeping flora, as well as rich growing environments, such as aquatic, moist and terrestrial.Lens [32] discovered significant variation in the stems of Impatiens species and explored the evolutionary relationship between herbaceous and woody forms in Impatiens by studying their anatomical structure.Stem plays an important role in the growth and development of plants.Stem xylem is related to plant growth rate and drought adaptation [33][34][35].Słupianek [36] found that the differences in the vessel diameter in the secondary xylem of Salix polaris stems is mainly related to the humidity of the habitat.These researches highlight the significant role that stems play in plant adaptation to their environment.The diversity among stems of Impatiens species provides excellent resources for studying the origin and evolutionary adaptation of Impatiens to different environments.Impatiens uliginosa thrives in aquatic regions, with upright and hollow stems.Impatiens chlorosepala is born in the moist area, it has small and solid fleshy stems that creep as they grow.Impatiens rubrostriata grows in sparse forests or shrub grasslands with solid stems and relatively high lignification.
In this study, I. uliginosa, I. rubrostriata and I. chlorosepala were used as experimental materials.We aimed to analyze the differences in stem tissue among these three Impatiens species with varying lignification degrees in different habitats.This was achieved through morphological anatomy analysis, as well as the determination of stem lignin content and composition, MYB4 gene cloning, gene sequence structure, systematic evolution, and qRT-PCR analysis.This study can not only contributes to understanding the adaptation mechanism of Impatiens species to different habitats but also provide essential data and theoretical support for investigating the evolutionary mechanism of Impatiens species from aquatic to terrestrial environments, because lignin has an important influence on the evolutionary process of plants from aquatic to terrestrial [37,38].Furthermore, it helps to provide basic data and theoretical support for the study of the adaptation mechanism of Impatiens to aquatic and terrestrial habitats, and is conducive to further improving the characteristics and resistance of Impatiens through the use of genetic engineering and other technologies, to promote the development and utilization of Impatiens and enrich the garden landscape.

Experimental materials
Three Impatiens species were collected from the field and planted in the greenhouse of Southwest Forestry University arboretum to simulate the growing environment for routine water and fertilizer management.I. chlorosepala grows in wet forests and streams and has creeping stems, I. uliginosa grows well in water and has upright stems, I. rubrostriata thrives in dry hillside rock and also has upright stems (Fig. 1).The anatomy of stem morphology selected the stem base of 5-10 cm above the ground that grows strong after flowering and maturity for the experiment.Lignin content was determined using whole stems of three Impatiens species.The cloning of the MYB4 gene was utilized to select healthy plant stems, which grow until flowering and maturity.The healthy root tip (5 cm), the stem base 5 cm above the ground, and the fourth mature leaf from top to bottom were selected for qRT-PCR.

Histochemical analysis
The paraffin sectioning method was modified based on Tong et al. [39].The collected samples were fixed in FAA solution and then dehydrated by alternating 70%, 85%, 95%, 100% ethanol, and 1/2 absolute ethanol + 1/2 pure xylene (v/v).After washing with xylene, the stems were embedded in paraffin.Paraffin sections of 10 μm were cut with a slicer, dried and stained with saffron fixation, sealed with gum, observed and photographed with the Eclipse Ci-L microscope.The anatomical structures of three Impatiens species stems were determined using Caseviewer software.The corresponding structural indices of the main determination are listed in Table S1.

Determination of total lignin content
The stems of three Impatiens species were dried in an oven at 37 ℃, weighed with an analytical balance until the stems dried to a constant weight, ground into fine powder with a mortar, sieved with a 40-mesh sieve and stored in a dryer for later use.The lignin content consisted of acid-soluble and acid-insoluble lignin, and the specific determination method referred to the method of Wu et al. [40].
0.5000 g (W1) of dried stem tissue that has passed through a 40-mesh sieve was weighed.The sample was extracted with benzene-ethanol (67:33, v/v) in a Soxhlet extractor for 4 h.After air drying, the powder was transferred into a 250.0 mL triangular flask and treated with 72% concentrated sulfuric acid at 30℃ and 120 r/min for 1.5 h.The hydrolysate was filtered using a G3 crucible filter.The residue was washed with distilled water until it reached a neutral pH.The filtrate was then adjusted to a volume of 250.0 mL and the concentration of sulfuric acid was 2.88%.The residue and filter were dried at 80℃ until a constant weight was achieved (W2).The filter and residue were placed on a scale and transferred to a muffle furnace.The mixture was heated at 200℃ for 30 min and then ashed at 575 ± 25℃ for 4 h.The mixture should be removed and allowed to cool in a dryer for 30 min.Weigh the sample to obtain W3 (ash + filter). 1 mL of the 250.0 mL filtrate was taken and diluted ten times with 2.88% sulfuric acid (depending on the sample).The absorbance was measured at 205 nm using an ultraviolet spectrophotometer, ensuring that it fell between 0.2 and 0.7.The dilution multiple was recorded as D. The control used was 2.88% sulfuric acid.Repeat each sample in triplicate.

Lignin monomer content and composition analysis
The material treatment method was identical to that used to determine the lignin content.Alkaline nitrobenzene oxidation method was used to determine the lignin monomer content in the stems of three Impatiens species by high-performance liquid chromatography.The specific steps were taken from Xu et al. [41].
0.1000 g of stem tissue powder was weighed (passed through a 40 mesh sieve), extracted for 6 h in a Soxhlet extractor containing benzene/ethanol (67:33, V/V) solution and air dried to constant weight to obtain cell wall residue (CWR).Weigh 50.0 mg of CWR, place in a sealed PTFE tank (25.0 mL) and add 5.0 mL of 2.0 mol/L NaOH solution, 0.5 mL of nitrobenzene and a rotor.After tightening and sealing the whole apparatus, it was placed in a constant temperature oil bath at 170℃ for 3.5 h at a rotation speed of 15 r/ min.After the reaction, the sealed vessel was rapidly cooled with cold water.The reaction mixture in the sealed vessel was transferred to a 100.0 mL triangular flask.Add a certain amount of internal standard to the triangular flask, the internal standard is ethyl vanillin and the concentration is 4.0 mg/ mL (2.0 mol/L NaOH configuration).The reaction mixture was extracted three times with 30.0 mL of dichloromethane/ethyl acetate mixture (1:1, V/V).Retain the aqueous phase (in this step the upper layer of the extract is retained and the lower layer contains nitrobenzene derivatives).Adjust the pH of the aqueous phase to 3-4 with 6.0 mol/L HCl and shake well.Extract again three times with 30.0 mL dichloromethane/ethyl acetate mixture (1:1, V/V).This step retains the bottom layer of the extract.The organic phase was collected and evaporated at 40℃ to obtain a solid residue.The residue was redissolved with 5.0 mL of chromatographically pure methanol, filtered through a 0.22 µm membrane filter, and 20.0µL was taken for HPLC detection.

Gene cloning
Screen MYB4 genes from three Impatiens species transcriptome data and carry out subsequent experiments.Total RNAs were extracted from mature stems of three Impatiens species using the Plant RNA Kit R6827 of Omega Bio-Tek, and then the first strand of cDNAs were synthesized using the TransScript ® One-Step gDNA Removal and cDNA Synthesis SuperMix of TransGen Biotech.Using first strand of cDNAs as templates, three Impatiens species' full-length MYB4 genes were amplified.The primers used are indicated in Table S2.The PCR reaction system consisted of 10 × EasyTaq Buffer(Mg 2+ ) 2.4 μL, dNTP 1.6 μL, EasyTaq DNA Polymerase 0.2 μL, upstream and downstream primers 1 μL each, cDNA template 1 μL, ddH 2 O 12.8 μL, total volume of 20 μL.The PCR reaction procedure is: 95℃ 5 min; 95℃ 50 s, 56℃ 30 s, 72℃ 1 min, 35 cycles; 72℃ 10 min; 4℃ 10 min.
According to the DNA extraction kit DP3111 of Bioteke, the DNAs of mature stems of three Impatiens species were extracted and the genomic DNAs of MYB4 genes of three Impatiens species were cloned using them as templates.The PCR reaction system and procedure were identical to those described above.

Fluorescence quantitative analysis
Total RNAs were extracted from the roots, stems, and leaves of three Impatiens species.Actin gene was used as the reference gene.Primers for qRT-PCR analysis were designed according to the obtained MYB4 sequences of three Impatiens species.Primers of structural genes COMT, F5H, C3H and HCT in three Impatiens species were designed in highly conserved fragments, as show in Table S3.
Using three-step qRT-PCR, the expression levels of different copies of the MYB4 gene in roots, stems and leaves of three Impatiens species were detected to analyze.According to the expression levels of different copies of MYB4 gene in different parts of Impatiens, the copies with strong function in the stems of three Impatiens species were screened, and the expression levels of MYB4 gene in three Impatiens species were analyzed using universal reference primers and the expression levels of structural gene in the stems of three Impatiens species were also analyzed.The reaction system of qRT-PCR is: qPCR SYBR Green Master Mix (Yisheng Company) 10 μL, ddH 2 O 8.2 μL, F primer 0.4 μL, R primer 0.4 μL, cDNA 1 μL, total volume 20 μL.Amplification procedure: 95℃ 5 min; 95℃ 15 s, 60℃ 30 s, 72℃ 1 min, 40cycles; 95℃ 15 s; 60℃ 1 min; 95℃ 15 s.For cloning and the relative expression levels data of the MYB4 genes from I. chlorosepala, see Li et al. [42].

Structural composition of stems of three Impatiens species
The anatomical structures of the stems of three Impatiens species are basically the same, consisting of epidermis, cortex, phloem, cambium, xylem, parenchyma, and pith from outside to inside (Fig. 2 A, D, G).The epidermis of three Impatiens species is composed of one layer of cells.While I. uliginosa and I. rubrostriata have regularly arranged and neatly organized epidermal cells, I. chlorosepala has twisted and irregular epidermal cells (Figure S1).The cortex is composed of parenchyma cells and has multiple layers of cells.The cortex lies adjacent to the xylem and phloem, and the xylem of I. chlorosepala is smaller, and the vascular bundle area is not obvious.The vascular bundles of I. uliginosa and I. rubrostriata are clearly visible, with obvious xylem, and relatively larger vessel diameter.The parenchyma cells within the pith of three Impatiens species are of large size.The arrangement of parenchyma cells in the pith of I. chlorosepala is disordered, while fewer parenchyma cells exist in the pith of I. uliginosa and the middle part of its pith is hollow, I. rubrostriata boasts a close and orderly arrangement of parenchyma cells (Fig. 2 B, E, H; Figure S2).
The cells of I. chlorosepala are irregular, arranged in disorder and loose.However, the cells of I. uliginosa are mostly regular square and closely arranged.I. rubrostriata cells are mostly elongated, which are closely arranged layer by layer from epidermis to xylem, and are more Fig. 2 Transverse (A, B, D, E, G, H enlarged in the pith parenchyma (Fig. 2 A, B, D, E, G,  H).The stem composition of three Impatiens species is nearly identical, while the cell shapes differ significantly among them.Additionally, all three Impatiens species have scalariform vessels (Fig. 2 C, F, I).

Impatiens species
The epidermis of I. uliginosa is the thickest, which is significantly different from I. chlorosepala and I. rubrostriata.Additionally, I. uliginosa has the largest stem diameter among the three species at the same growth stage.The thickness of cortical cells, epidermis, vessel diameter, pith diameter, xylem thickness, phloem thickness, and cell wall are also significantly different from I. chlorosepala and I. rubrostriata.All other indicators do not show any noticeable differences between the species.The above results may be attributed to the stem diameter of Impatiens species.
However, among three Impatiens species, the ratio of cell wall thickness to stem diameter (Cw/D), ratio of xylem thickness to stem diameter (Xyl/D), ratio of vascular bundle area to stem cross-sectional area (Vb/S) and ratio of xylem area to stem cross-sectional area (Sxyl/S) are the highest in I. rubrostriata compared to I. uliginosa and I. chlorosepala, which is a significantly distinction among the three Impatiens species.The results show that the lignification degree of the erect semi-woody I. rubrostriata is indeed higher than that of the erect I. uliginosa and the creeping I. chlorosepala, indicating a strong correlation between the lignification degree of Impatiens species and their stems thickness, cell wall thickness, as well as the ratios of vascular bundle area to stem crosssectional area and xylem area to stem cross-sectional area (Table 1).

Correlation of stem morphology and anatomical structure of three Impatiens species
The results of the correlation analysis indicate that the stem diameter of I. chlorosepala has a positive correlation with both cell wall thickness and xylem area, as well as a negative correlation with all other indexes.Additionally, xylem thickness is positively correlated with Xyl/D and Sxyl/S, the cell wall thickness is positively correlated with Cw/D, and the vascular bundle area is positively correlated with Vb/S and Sxyl/S (Table S4).In I. uliginosa, stem diameter exhibits a positive correlation with xylem area and vascular bundle area, and a negative correlation with Cw/D, Xyl/D, Vb/S and Sxyl/S.Xylem thickness demonstrates a significant positive correlation with Xyl/D, while cell wall thickness shows a positive correlation with Cw/D and Sxyl/S (Table S5).In I. rubrostriata, the stem diameter is negatively correlated with Vb/S and Sxyl/S, the cell wall thickness is positively correlated with Cw/D, the xylem thickness is positively correlated with Cw/D and Sxyl/S, and Sxyl/S is positively correlated with Cw, Sxyl, Vb, Cw/D, Xyl/D and Vb/S (Table S6).We speculate that among the three Impatiens species, the stem diameter may have a certain influence on the anatomical structure of the stem, but Sxyl/S is mainly related to the xylem thickness, vascular bundle area and cell wall thickness.

Impatiens species
The lignin content of three Impatiens species stems is significantly different.The acid-soluble lignin content of I. chlorosepala is 3.6%, that of I. uliginosa and I. rubrostriata is 9.78% and 9.46% respectively, which is significantly different from that of I. chlorosepala.The acid-insoluble lignin content in three Impatiens species is comparatively low, among which I. chlorosepala and I. uliginosa have similar values at 0.61% and 0.65%, respectively, while I. rubrostriata has a significantly higher content of 1.27% (Table 2).The highest content of total lignin was I. rubrostriata (10.73%), followed by I. uliginosa (10.43%) and I. chlorosepala (4.21%).Among the three Impatiens species, the erect semi-woody I. rubrostriata has the highest lignification degree, while the creeping herb I. chlorosepala has the lowest lignification degree.These results are consistent with the findings of stem anatomy analysis.

Analysis on the difference of lignin monomer content in the stem of three Impatiens species
The determination of lignin monomer content of three Impatiens species shows that I. chlorosepala has the lowest levels of H and G-lignin and lacks S-lignin, which may be related to its creeping growth habit.There is no significant difference in S-lignin between I. uliginosa and I. rubrostriata, but H-lignin and G-lignin of I. rubrostriata are significantly higher than I. uliginosa.These findings further support the conclusion that I. rubrostriata has the highest degree of lignification in its stems (Table 3; Figure S3).

Correlation analysis between total lignin content and morphological anatomy of three Impatiens species
Through the correlation analysis between the lignin content and the anatomical morphological indexes of three Impatiens species stems, it was found that the lignin content was positively correlated with the anatomical structure of stems, and the correlation coefficient with xylem thickness, cell wall thickness and Cw/D was higher (Table S7).This implies that the higher the Sxyl/S in morphological anatomy, the higher the lignin content and the lignification degree of Impatiens species' stems.

Cloning and sequence analysis of MYB4 gene from three Impatiens species
Three copies of MYB4 genes are present in I. chlorosepala and I. uliginosa, whereas only two copies are found in I. rubrostriata.The target band for the gene is 600-900 bp, and they were named as IcMYB4-1, IcMYB4-2, and IcMYB4-3 for I. chlorosepala, IuMYB4-1, IuMYB4-2, and IuMYB4-3 for I. uliginosa, IrMYB4-1 and IrMYB4-2 for I. rubrostriata.The genomic DNA bands of MYB4 genes in three Impatiens species range from 700-1000 bp (Table S8; Figure S4, S5, S6).Using the Expasy website (http:// expasy.org/ tools/), the amino acid sequences encoded by MYB4 genes in three Impatiens species were predicted.The results indicate that all MYB4 proteins are hydrophilic and unstable (Table S9), and all MYB4 proteins are mainly located in the nucleus (Table S10).The MYB4 proteins of three Impatiens species all contain the SANT domain and the DNA binding site of MYB, indicating that they belong to  the MYB family.Moreover, the MYB4 proteins also contain a stable PLN03091 domain, which is basically consistent with the conserved domain of MYB4 proteins in other plants.Hence, it is speculated that the MYB4 genes of three Impatiens species belong to the R2R3-MYB subfamily (Figure S7, S8, S9).Multi-sequence comparison analysis of MYB4 proteins of three Impatiens species and other species revealed high conservation of all genes within the MYB familyspecific domain.There are conserved R2 and R3 DNA binding domains at the N-terminal, but there are considerable variations at the C-terminal.Additionally, the inhibitory conserved motif EAR is present (Figure S10).
The phylogenetic tree was constructed by NJ method with the MYB4 protein of three Impatiens species and its homologous MYB4 amino acid sequence, using MEGA7.0software.The results show that the phylogenetic tree is divided into three branches.Three MYB4-1 genes of I. chlorosepala, I. uliginosa, and I. rubrostriata cluster on a small branch, indicating a closer genetic relationship.I. rubrostriata and I. chlorosepala's MYB4-2 get together, which is supposed to be a orthologous relationship.These genes are grouped into a big branch with plants including Actinidia rufa, Salvia splendens, Salvia hispanica, Vitis vinifera, Carya illinoinensis, and Perilla frutescens.I. uliginosa's IuMYB4-2 and IuMYB4-3 are grouped together in another branch, which is also supposed to be a orthologous relationship.However, IcMYB4-3 from I. chlorosepala is clustered into a small branch alone, and it was conjoined with Rosa chinensis and Benincasa hispida, indicating orthologous.The above genes and plants such as Camellia sinensis and Vitis davidii are grouped into the second branch.The third branch comprises Angelica sinensis, Salvia miltiorrhiza, Solanum stenotomum and Capsicum chinense, all of which are annual or perennial herbs (Fig. 3).According to the phylogenetic tree, it can be inferred that the MYB4 genes in three Impatiens species are closely related to those in some herbs and woody plants.Furthermore, they may have similar functions with the genes in other plants that are grouped together.

Expression analysis of MYB4 gene in three Impatiens species
MYB4 was expressed in the stems of three Impatiens species.The expression level was highest in I. chlorosepala, which had the lowest lignin content, followed by I. uliginosa, and the expression level in I. rubrostriata with the highest lignin content was the lowest (Fig. 4D).Combined with the determination and anatomical data on the lignin content in the stems of three Impatiens species, it is suggested that MYB4 gene may has a negative regulatory impact on the synthesis of lignin in Impatiens.
Three IcMYB4 genes were expressed in the roots, stems, and leaves of I. chlorosepala (Fig. 4A).IcMYB4-1 had the lowest expression level in the stem of I. chlorosepala, followed by the leaf, and the highest expression level in the root.IcMYB4-2 had the highest expression in leaves, about eight times that in stems, and the lowest expression in roots.The expression trend of IcMYB4-3 was consistent with that of IcMYB4-2.Among the three copies of I. chlorosepala, IcMYB4-1 has the highest expression in the root, followed by IcMYB4-2 and IcMYB4-3, indicating that IcMYB4-1 exerts a more dominant function within the root of I. chlorosepala.Furthermore, IcMYB4-2 has the highest expression level in the leaves, which suggests that IcMYB4-2 primarily functions in the leaves of I. chlorosepala.Lastly, IcMYB4-3 demonstrates the highest expression in the stem compared to the other two copies, indicating its main function in the stem of I. chlorosepala.In three different parts of I. chlorosepala, the lignification degree of roots is relatively high, with more lignin, but less lignin in leaves.It is speculated that IcMYB4-1 gene may positively regulate lignin synthesis in I. chlorosepala, while IcMYB4-2 and IcMYB4-3 may have a negative effect on lignin synthesis.The study indicates that there were some differences in the mechanism of different copies of IcMYB4 genes on lignin synthesis regulation in different parts of I. chlorosepala.
Three IuMYB4 genes were expressed in the roots, stems, and leaves of I. uliginosa.The expression patterns of IuMYB4-1, IuMYB4-2 and IuMYB4-3 genes were consistent across different parts of I. uliginosa, with the lowest expression in the stems, the second in the roots and the highest expression in the leaves.It is speculated that all three copies have an important function in the leaves of I. uliginosa (Fig. 4B).The expression level of IuMYB4-2 is the lowest among the three copies, indicating that it may have the least significant role in I. uliginosa.The expression level of IuMYB4-3 in three different parts of I. uliginosa is highest among the three copies, and it is speculated that IuMYB4-3 plays the strongest role in I. uliginosa.Three copies of IuMYB4 of I. uliginosa are all the highest in the leaves, which contain the least lignin content.Despite the differences in their possible functions in various parts, they all play a similar role and negatively regulate lignin synthesis in I. uliginosa.
The expression of IrMYB4-1 in the root of I. rubrostriata was the lowest, followed by the stem, and the highest expression was in the leaves, indicating that IrMYB4-1 played a stronger role in the leaves of I. rubrostriata.IrMYB4-2 displayed the lowest expression level in stems and the highest in leaves, implying that both IrMYB4-1 and IrMYB4-2 have great influence on lignin synthesis in leaves of I. rubrostriata (Fig. 4C).The expression levels of IrMYB4-1 in three different parts of I. rubrostriata were lower than that of IrMYB4-2, which indicated that IrMYB4-2 plays a stronger role in I. rubrostriata.The expression level of IrMYB4 in the root and stem of I. rubrostriata with high lignification is low, but it is high in the leaves with low lignification, which indicates that two copies of IrMYB4 could be involved in negative regulation of lignin synthesis in I. rubrostriata.However, there may be some differences in both the intensity and mechanism between the different copies.

Expression analysis of structural genes related to lignin biosynthesis in three Impatiens species
Structural genes involved in lignin synthesis, such as F5H, C3H, COMT, and HCT, were all expressed in the stems of three Impatiens species (Fig. 5).Notably, F5H expression was highest in I. rubrostriata, followed by I. uliginosa, while I. chlorosepala had the lowest expression, suggesting that F5H has a greater effect on I. rubrostriata.The expression of C3H was highest in I. rubrostriata, followed by I. chlorosepala, and lowest in I. uliginosa, indicating a major role of C3H in the stems of I. rubrostriata.In contrast, the expression of COMT was the lowest in the stems of I. chlorosepala, followed by I. rubrostriata, and the highest in I. uliginosa, indicating that COMT may play a stronger role in the stems of I. uliginosa.HCT expression is absent in I. chlorosepala, which means that it does not affect on the stem of I. chlorosepala, and its expression in I. rubrostriata is higher than that in I. uliginosa, so HCT mainly plays a functional role in I. rubrostriata.
The stem lignin content of I. rubrostriata is the highest among the three Impatiens species, followed by I. uliginosa, with I. chlorosepala having the lowest content.The F5H, C3H, and HCT genes were most expressed in I. rubrostriata's stems, while COMT was most expressed in the stems of I. uliginosa.F5H, HCT, and COMT were least expressed in I. chlorosepala's stems, with C3H being least expressed in I. uliginosa's stems.The expression levels of the aforementioned structural genes are lower in I. chlorosepala, but high in I. rubrostriata, which is contrary to the expression trend of MYB4 gene in three Impatiens species.It can be seen that MYB4 genes may inhibit the expression of F5H, HCT, and COMT in I. chlorosepala, resulting in the lowest lignin content in its stems and the absence of S-lignin.However, in the I. rubrostriata stem, the expression of MYB4 gene is the lowest, resulting in the weakest inhibition on its structural gene and it may only a partial effect on COMT, so its lignin content is the highest.These results suggest that transcription factors such as MYB4 may regulate structural genes such as F5H, C3H, COMT, and HCT, and have certain influence on the lignin content of Impatiens species' stems.

Discussion
The lignification degree of stems of three Impatiens species may be closely related to their growing environment and morphology During plant evolution, they adapt to the environment and some physiological changes through a series of morphological and structural alterations.For instance, the anatomical structure changes of stems can enhance their ability to tolerate cold, resist drought and lodging, as well as determine their suitability for the aquatic or terrestrial environment [43].
The stems of three Impatiens species are composed of several layers, including the epidermis, cortex, phloem, cambium, xylem, and pith.Studies have demonstrated that plants with more developed pith and an increased number of vessels possess a greater capacity to store water [44,45].Among the three Impatiens species, I. chlorosepala predominantly grows in wet forest areas along streams, and the pith cells are disordered and loosely arranged.I. uliginosa mostly grows in water and has a hollow pit.I. rubrostriata thrives in dry hillside woodland, with closely arranged and well-developed pith cells.I. rubrostriata has more vessels compared to I. uliginosa, while I. chlorosepala has the least number of vessels.We speculate that I. rubrostriata, which grows in arid hillside woodland, may require a greater water supply during growth, which leads to more developed stem pith and more vessels.
The stem diameter of I. uliginosa is the largest, and the thickness of cortical cells, vessel diameter, pith diameter, xylem thickness, and phloem thickness are all larger than those of the other two Impatiens species.Previous studies have shown that the thickness of cortical cells, vessel diameter, xylem thickness and phloem thickness can affect a plant's water transport and energy storage of plants [46,47].The above indexes of I. uliginosa are higher than those of I. rubrostriata grown in arid areas, possibly due to differences in the shape and growth speed of the plant itself.I. uliginosa can grow up to 1 m per year, whereas I. rubrostriata reaches only around 0.5 m.Among the three Impatiens species, the cell wall of I. uliginosa is the thickest, while that of I. chlorosepala has nearly negligible cell wall.This difference may be attributed to the larger plant height and stem thickness of I. uliginosa as compared to I. chlorosepala and I. rubrostriata during the same growth period.The ratio of xylem area to stem cross-sectional area is the highest in I. rubrostriata, reaching 11.4%, I. uliginosa follows with 6.58%, and I. chlorosepala with 5.48%, which is consistent with the research results of Chen [48] the higher the ratio of xylem area to stem cross-sectional area, the higher the lignification degree of stems.
According to the aforementioned findings, one can deduce that the lignification degree of plants may be related to their growing environment and morphology.The stems of I. chlorosepala grow in a creeping manner, resulting in weak mechanical support, thus leading to a thin cell wall and xylem and a low the ratio of xylem area to stem cross-sectional area.The degree of lignification in I. uliginosa with erect stems is higher than that of I. chlorosepala with creeping stems.However, the lignification degree of I. rubrostriata with the same upright stem is greater than that of I. uliginosa with upright stem, suggesting a potential correlation between lignification and plant growth environment.Plants in an aquatic environment exhibit a lower degree of lignification, whereas those in a terrestrial environment exhibit a higher degree.For example, Wang's [49] research found that Alternanthera philoxeroides exhibits higher lignification levels in terrestrial environments compared to aquatic habitats.Nevertheless, the lignification degree of plants is not only related to the morphological anatomy of stems, but also related to the lignin content in stems.Thus, a comprehensive examination of stem lignification from various perspectives is necessary.

Comparative analysis of lignin content and composition in stems of three Impatiens species
To support our results on stem morphology, we analyzed the lignin content and lignin monomer content in three Impatiens species.Lignin is a secondary metabolite that plays a crucial role in plant growth, development, and response to adverse environmental stress.The concentration of lignin varies not only between different species but also within the same species at different growth stages.Typically, coniferous wood has a lignin content of 27-33%, while broadleaf wood and herbs have 18-25% and 17-24%, respectively [50].There is a positive correlation between the total lignin determination and the morphological anatomy of the stem.The total lignin content of I. chlorosepala is 4.21%, which differs significantly from I. uliginosa and I. rubrostriata.The variation in total lignin content among three Impatiens species may be connected to their upright or creeping growth habits.
Studies have shown that S-lignin is related to the mechanical strength and vertical growth of plants [51].In the determination of lignin monomer content, I. chlorosepala lacks S-lignin, and there is no significant difference in S-lignin between I. uliginosa and I. rubrostriata, which is consistent with the morphological characteristics of the creeping growth of stems of I. chlorosepala and the vertical growth of I. uliginosa and I. rubrostriata.The H-lignin and G-lignin of I. rubrostriata were significantly higher than those of I. uliginosa, while I. chlorosepala showed the lowest value.This finding provides evidence that the lignification degree of I. rubrostriata with upright stems growing in arid hillside woodlands is the highest, and that of I. uliginosa with erect stems in water is higher than that of I. chlorosepala with creeping stems in wet forests.The results show that the lignification degree of plant stems may be closely related to their growth characteristics and lignin content.

The regulatory role of MYB4 gene in lignin biosynthesis of three Impatiens species
The MYB4 genes from three Impatiens species that we isolated and cloned belong to the MYB family.Many researches show that theR2R3-MYB family has the N-terminal amino acid motif that is relatively more conservative and the C-terminal amino acid motif that is extremely low [52,53].It is consistent with the results of this study, the DNA domain of MYB transcription factors is highly conserved across species.Phylogenetic analysis indicates that the MYB4 genes in three Impatiens species share a close evolutionary relationship with plants including Camellia sinensis, Actinidia rufa and Vitis vinifera.CsMYB4 in Camellia sinensis [54], and VvMYB4b in Vitis vinifera 'Moldova' [55], have both been shown to inhibit lignin synthesis, and it is possible that the MYB4 genes in three Impatiens species also play a negative regulatory role in lignin synthesis.The MYB4 gene's copy number varies among plant species, and they are expressed in all parts of most plants.Even within the same species, the expression level significantly varies among different plant parts.For instance, CsMYB4-5 and CsMYB4-6 exhibit high expression in roots and low expression in stems in Camellia sinensis [54].In this study, the expression levels of MYB4 genes were lower in roots or stems and higher in leaves of three Impatiens species.This trend is consistent with the observed expression pattern observed in other plants.We speculated that the MYB4 transcription factors may have transcriptional inhibition.
Research has demonstrated that overexpression of the TaMYB4 transcription factor in wheat leads to a decrease in total lignin content [56].In Arabidopsis, MYB4 suppresses the expression of genes related to lignin [18].The studies demonstrate that the MYB4 gene inhibits the regulation of lignin synthesis.However, the overexpression of the PbrMYB4 gene in pear calli and Arabidopsis thaliana promotes lignin accumulation in Pyrus bretschneideri fruit stone cells.This provides evidence that the PbrMYB4 gene has a positive regulatory effect on lignin biosynthesis in pear fruit stone cells [57].It indicates that the regulation of the MYB4 gene on lignin may vary among different plants.In this study, it was also found that IcMYB4-1 exhibited the highest expression in the root of I. chlorosepala, while having low expression in the stem and leaf, suggesting that it may exert a positive influence on regulating lignin synthesis in I. chlorosepala.The two other IcMYB4 genes show the highest expression levels in leaves, but the expression levels are relatively low in roots and stems with high lignification, so it is speculated that they may play a negative regulatory role in lignin synthesis of I. chlorosepala.The expression levels of MYB4 of I. uliginosa and I. rubrostriata are low in roots and stems with high lignin content, and high in leaves with low lignin content.Further verification is needed to determine the regulatory mechanism and function of the MYB4 genes in the regulation of lignin synthesis in Impatiens species, as indicated by the aforementioned findings.
The expansion and contraction of genes play a crucial role in the adaptive evolution of plants [58,59].For example, in four Poaceae species (rice, maize, sorghum, and brachypodium), NBS genes showed a contraction evolutionary pattern [58,60].In the study on Orchidaceae, Wang et al. [61] found that CslA, a gene related to plant cell wall polysaccharide synthesis with expansion, may play a key role in the response of different life forms of Orchidaceae (epiphytic, terrestrial, and saprophytic) to drought stress.This study found that aquatic I. uliginosa and wet I. chlorosepala have three MYB4 genes, while terrestrial I. rubrostriata has only two MYB4 genes.Whether this change in the number of MYB4 genes between Impatiens species is related to plant adaptation to the environment and/or the growth state of the plants themselves.It is also unclear whether the gene expansion from terrestrial to aquatic environments or gene contraction from aquatic to terrestrial environments is responsible.Additionally, while MYB4 negatively regulates lignin biosynthesis, but the expression of IcMYB4-1 in I. chlorosepala is positively correlated with lignin content.Further study is required to determine whether gene expansion during adaptive evolution leads to the functionalization of genes.

MYB4 negatively regulates lignin biosynthesis by inhibiting the expression of structural genes
MYB4 regulates lignin synthesis by controlling the expression of structural genes in lignin synthesis pathway.Yu [24] found that lignin-related genes CCR and C4H were down-regulated in birch that overexpressed BpMYB4, CAD and C4H genes were up-regulated in birch which suppressed BpMYB4, which indicates that BpMYB4 transcription factor can negatively affect the expression of some lignin biosynthesis genes compared to wild type.When PvMYB4 was overexpressed in Panicum virgatum, the expression levels of structural genes in transgenic plants decreased obviously, including COMT, C3H, F5H, and HCT [22].This research shows that MYB4 has the ability to inhibit the expression of structural genes.In this study, the expression levels of structural genes F5H and HCT were the lowest in I. chlorosepala and the highest in I. rubrostriata, which contradicts the expression trend of MYB4 in the stems of three Impatiens species.It was speculated that MYB4 could inhibit the expression of the two structural genes.Additionally, MYB4 expression was the highest in the stem of I. chlorosepala, resulting in the lowest expression of C3H and COMT among the three Impatiens species.The expression levels of C3H and COMT are high in I. rubrostriata.It is hypothesized that the low expression level of MYB4 in I. rubrostriata leads to a feeble inhibitory effect on the two structural genes.
The synthesis process of lignin monomers can be regulated by reducing the activity of structural genes.Overexpression of the F5H structural gene in plants results in the production of a large quantity of S-lignin in the plants [62,63].COMT regulates G-lignin, but has the capability of also regulating S-lignin [64].C3H and HCT heavily influence H-lignin [65,66].I. chlorosepala lacks S-lignin, so it is consistent with the lowest expression level of F5H in the stems of I. chlorosepala.Moreover, the H-and G-lignin of I. chlorosepala are lower than these of I. uliginosa and I. rubrostriata, there is no HCT expression in I. chlorosepala, but the expression of C3H is higher than that of I. uliginosa.It is speculated that the H-lignin of I. chlorosepala is mainly generated by C3H catalysis.The three lignin monomers of I. uliginosa are all lower than those of I. rubrostriata.Additionally, the expression of other structural genes, except for COMT, is higher in I. rubrostriata, which may be attributed to the fact that COMT is also involved in the synthesis of S-lignin in I. uliginosa.Among the three Impatiens species, I. rubrostriata exhibits the highest content of three lignin monomers, and the expression of its structural genes positively correlates with the lignin monomer content.The synthesis of lignin monomer is supposed to be controlled by the structural genes in the stems of three Impatiens plants regulated by MYB4 transcription factors, thus affecting the lignin content and the lignification degree of Impatiens species.However, the lignin synthesis pathway is completed through multi-gene and multi-level coordination; therefore, further exploration is necessary to understand the specific pathway of lignin synthesis in Impatiens.

Conclusion
There are significant differences in anatomical parameters among the stems of three Impatiens species.Moreover, the stems of I. rubrostriata exhibit the highest content of lignin and lignin monomer, followed by I. uliginosa and I. chlorosepala.The anatomical structure is consistent with the determination results of lignin content.I. rubrostriata has a higher degree of lignification due to its upright and solid stems, while I. uliginosa exhibits a lower degree of lignification with its upright and hollow stems, and I. chlorosepala displays the lowest degree of lignification owing to its creep and solid stems.
Three copies of MYB4 genes in I. uliginosa and I. chlorosepala and two copies of I. rubrostriata were isolated and cloned.The MYB4 proteins in three Impatiens species demonstrate high similarity with other plants.They possess conserved R2 and R3 DNA binding domains at the N-terminal and demonstrate a high homology.In addition, they have an inhibitory conserved motif EAR at the C-terminal.Except for IcMYB4-1, which was the highest expression level in the root of I. chlorosepala, and the other MYB4 genes of three Impatiens species were mainly expressed in the leaves, with the stems and roots following behind.This suggests the potential for disparities between the different copies of the MYB4 gene within the three Impatiens species.According to the lignin content and the differential expression of MYB4 and structural genes in the stems of three Impatiens species, it is speculated that MYB4 negatively regulates the lignin synthesis in the stems of three Impatiens species by regulating the expression of structural genes, and its regulation mechanism appears to vary across different Impatiens species.

Fig. 4
Fig. 4 The expression pattern of MYB4 in different parts of three Impatiens species.A The expression pattern of MYB4 in different parts of I. chlorosepala.B The expression pattern of MYB4 in different parts of I. uliginosa.C The expression pattern of MYB4 in different parts of I. rubrostriata.D Relative expression of MYB4 gene in the stems of three Impatiens species.Different lowercase letters indicate significant differences (P < 0.05)

Fig. 5
Fig. 5 Relative expression of structural genes in the stems of three Impatiens species.Different lowercase letters indicate significant differences (P < 0.05)

Table 1
Anatomical parameters of the stems of three Impatiens species

Table 2
Lignin content of the stems of three Impatiens species

Table 3
Lignin monomer content of the stems of three Impatiens species